Exposure of eukaryotic cells to ionizing radiation generates a spectrum of DNA lesions that trigger signaling cascades leading to DNA repair, cell cycle arrest, and in some cases apoptosis. Defects in these signaling pathways or in the repair machinery increase the probability of cell cycle progression in the presence of DNA damage, a condition linked to increased rates of oncogenic transformation in mammalian cells. This proposal addresses the function of the Mre1 1/Rad50/Nbs1 (MJRJN) complex, which plays a critical role in both cell cycle regulation and DNA repair following DNA damage, and acts specifically in response to DNA double-strand breaks. From experiments in yeast and in vertebrate cells we know that MJR/N is essential for non-homologous end joining and sister chromatid recombination, and also functions in meiotic recombination and telomere maintenance. In addition, mutations in the Mre11 and Nbs1 components of the complex are responsible for two rare chromosomal instability disorders in humans, Nijmegen Breakage Syndrome (NBS) and A-T-like-Disorder, which cause extreme radiation sensitivity, immunodeficiency, and high rates of malignancy. The clinical manifestations of these disorders are very similar to that of Ataxia-Telangiectasia (A-T), suggesting that MIR/N and the A-T-Mutated protein (ATM) function in the same DNA damage response pathway. In previous work we have established an expression system for the human M/R/N complex and have characterized the enzymatic activities of the complex extensively on model DNA substrates. In the work proposed here we will: A) test two models for the substrate specificity of the MJR/N nuclease in vivo and in vitro; B) determine the biochemical basis of defects associated with mutations in Mre1 1, Rad50, and Nbs1; and C) determine the biochemical consequences of Nbs1 phosphorylation by ATM, including effects on the enzymatic activities of the complex and associations with other proteins. These experiments will bridge the gap between our knowledge of the biochemistry of this complex and observations of the biological consequences of MJRJN mutations in yeast and in mammalian cells. This work will lead us toward our long-term goal of understanding at a mechanistic level how ATM and M/R/N function in DNA double-strand break repair as well as in meiotic recombination and telomere maintenance.